We investigate the molecular mechanisms that control the sorting of transmembrane proteins in the endosomal-lysosomal system. Sorting is mediated by interactions between signals in the cytosolic domains of the transmembrane proteins and adaptor protein associated with the cytosolic face of membranes. Two major types of sorting signals, referred to as tyrosine-based and dileucine-based, have been previously described. Work in this section has demonstrated that both types of signals are recognized with characteristic fine specificities by the adaptor protein (AP) complexes AP-1, AP-2, AP-3 and AP-4, or by the GGA adaptor proteins GGA1, GGA2 and GGA3. Mutations in AP-3 are the cause of the pigmentation and bleeding disorder, Hermansky-Pudlak syndrome type 2. Current work is aimed at elucidating the structure, regulation and physiological roles of the AP complexes and GGAs, and investigating the possibility that defects in these proteins underlie protein trafficking disorders.[unreadable] [unreadable] The characterization of the molecular machinery involved in protein sorting is important for the understanding of the pathogenesis of various metabolic and developmental disorders. Several years ago we discovered, in collaboration with William Gahl (NHGRI), that mutations in the beta3A subunit of AP-3 are the cause of the autosomal recessive disorder, Hermansky-Pudlak syndrome (HPS) type 2 (HPS2). This disease presents with reduced pigmentation and prolonged bleeding, which are due to abnormal melanosomes in melanocytes and absent dense bodies in platelets, respectively. HPS2 is thus a disorder of lysosome-related organelles that affects certain specialized cell types. The connection between the primary AP3 deficiency and the ultimate cellular and organismal phenotypes, however, was not well understood. In collaboration with and Alex Theos and Mickey Marks (University of Pennsylvania), and Graca Raposo (Curie Institute, Paris), we observed that AP-3 is associated with endosomes that are the precursors of melanosomes in melanocytes. In addition, we found that AP-3 deficiency prevents transport of tyrosinase, a key enzyme in the biosynthesis of the melanine pigment, from endosomes to melanosomes. Finally, we observed that AP-3 binds to a ?dileucine-based? signal (i.e., EKQPLL in humans; ERQPLL in mice) that is present in the cytosolic tail of tyrosinase. These findings indicate that AP3 is normally involved in the signal-mediated sorting of tyrosinase from endosomes to melanosomes, and that AP3 deficiency impairs this sorting, leading to the pigmentation defect that is characteristic of HPS2.[unreadable] [unreadable] Recognition of sorting signals by AP1 and GGAs leads to the incorporation of the signal-bearing cargo proteins into transport vesicles that bud from the trans-Golgi network (TGN) and subsequently fuse with endosomes. For a long time, these transport vesicles were thought to be uniformly spherical and to have a diameter of 60-100 nm, but recent fluorescent imaging of live cells showed that they actually have varying shapes and sizes (that is, they are ?pleiomorphic?). Analysis of the ultrastructure of these vesicles using correlative light-electron microscopy (CLEM) revealed the they range from typical 60-100nm coated vesicles to larger, convoluted tubular-vesicular structures that contain several coated buds. After detaching from the TGN, some of these pleiomorphic structures move long distances in the cytoplasm, until they eventually fuse with peripheral endosomes. We propose that pleiomorphic vesicles serve as vehicles for long-range distribution of biosynthetic or recycling cargo from the TGN to peripheral endosomes. These vesicles represent a novel type of coated vesicular intermediate involved in selective cargo transport between membrane-bound compartments.[unreadable] [unreadable] The AP complexes and GGAs have ?ear-like? domains that bind ?accessory proteins?. Most accessory proteins are thought to regulate the budding of transport vesicles and the incorporation of cargo proteins into these vesicles. However, work from our lab suggests that least one of these accessory proteins, the Rabaptin-5?Rabex-5 complex, regulates vesicle fusion of TGN-derived transport vesicles with endosomes. We previously showed that the Rabaptin-5?Rabex-5 complex does so through binding to the ear domains of the AP1 gamma subunit and of the GGAs via a canonical motif shared with other accessory proteins. Over the past year, we have found that the Rabaptin-5?Rabex-5 complex is regulated by ubiquitin. Mutational and structural analyses (the latter done in collaboration with James Hurley, NIDDK) showed that Rabex-5 has two binding sites for ubiquitin. The first site is a zinc finger (ZnF) that binds to a polar region centered on aspartate-58 of ubiquitin, whereas the second site is a new type of ubiquitin-binding domain, an alpha-helical, inverted ubiquitin-interacting motif (IUIM) that binds to a hydrophobic patch centered on isoleucine-44 of ubiquitin. This bipartite binding represents a novel mechanism of ubiquitin recognition that allows the formation of higher-order Rabex-5?ubiquitin structures. Mutation of ubiquitin-binding residues in Rabex-5 impairs its recruitment to endosomes and its ability to mediate endosome fusion, thus demonstrating an important role for ubiquitin in regulating the fate of transport vesicles.[unreadable] [unreadable] Newly-made lysosomal hydrolases are sorted by binding to mannose 6-phosphate receptors (MPRs) at the TGN. The hydrolase-receptor complexes are recognized by the GGAs, which mediate packaging into transport vesicles bound for endosomes. The acidic environment of endosomes induces the release of the hydrolases from the MPRs, after which the hydrolases follow the fluid phase to lysosomes, while the MPRs return to the TGN to mediate further rounds of transport. In previous work, we showed that the proteins, Vps29 and Vps35, which are subunits of a protein complex named ?retromer?, played a role in this retrograde transport of MPRs from endosomes to the TGN. We have recently examined the requirement of two other putative subunits of retromer, the sorting nexins 1 and 2 (SNX1 and SNX2). Using RNA interference, we found that depletion of either SNX protein by RNA interference had no effect on MPR trafficking, but combined depletion of both SNX proteins impaired the recycling of MPRs to the TGN and caused its missorting to lysosomes, where they were degraded. These findings demonstrated that SNX1 and SNX2 play interchangeable but essential roles, as part of the retromer complex, in the sorting of MPRs from endosomes to the TGN.[unreadable] [unreadable] To elucidate the structural bases for the role of retromer in MPR retrograde transport, we again collaborated with James Hurley (NIDDK) to solve the crystal structure of another retromer subunit, Vps26. The structure showed that Vps26 consists of two curved beta-sandwich domains (N and C) that are connected by a polar core and a flexible linker. This structure resembles that of arrestins, which are adaptor molecules involved in the internalization of and signaling by activated G-protein-coupled receptors. Arrestins are known to undergo dramatic conformational changes upon interaction with the phosphorylated receptor tails. The changes involve a reorientation of the two beta-sandwich domains such that they embrace the receptor tails through their concave surfaces. The resemblance to the arrestins suggests that Vps26 could undergo a similar conformational change in the process of recognizing the cytosolic tail of some transmembrane protein. Thus, Vps26 could be a cargo-recognition component of the retromer complex.